nanomaterials Article Polysaccharide-Based Bioink Formulation for 3D Bioprinting of an In Vitro Model of the Human Dermis Tanja Zidariˇc 1,* , Marko Milojevi´c 1, Lidija Gradišnik 1, Karin Stana Kleinschek 2,3,4, Uroš Maver 1,5,* and Tina Maver 1,2,* 1 Institute of Biomedical Sciences, Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia; [email protected] (M.M.); [email protected] (L.G.) 2 Laboratory for Characterization and Processing of Polymers (LCPP), Faculty of Mechanical Engineering, University of Maribor, Smetanova ulica 17, 2000 Maribor, Slovenia; [email protected] 3 Institute of Chemistry and Technology of Biobased Systems, Faculty of Technical Chemistry, Chemical and Process Engineering and Biotechnology, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria 4 Institute of Automation, Faculty of Electrical Engineering and Computer Science, University of Maribor, Koroška cesta 46, 2000 Maribor, Slovenia 5 Department of Pharmacology, Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia * Correspondence: [email protected] (T.Z.); [email protected] (U.M.); [email protected] (T.M.) Received: 10 February 2020; Accepted: 8 April 2020; Published: 11 April 2020 Abstract: Limitations in wound management have prompted scientists to introduce bioprinting techniques for creating constructs that can address clinical problems. The bioprinting approach is renowned for its ability to spatially control the three-dimensional (3D) placement of cells, molecules, and biomaterials. These features provide new possibilities to enhance homology to native skin and improve functional outcomes. However, for the clinical value, the development of hydrogel bioink with refined printability and bioactive properties is needed. In this study, we combined the outstanding viscoelastic behavior of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate (ALG), carboxymethyl cellulose (CMC), and encapsulated human-derived skin fibroblasts (hSF) to create a bioink for the 3D bioprinting of a dermis layer. The shear thinning behavior of hSF-laden bioink enables construction of 3D scaffolds with high cell density and homogeneous cell distribution. The obtained results demonstrated that hSF-laden bioink supports cellular activity of hSF (up to 29 days) while offering proper printability in a biologically relevant 3D environment, making it a promising tool for skin tissue engineering and drug testing applications. Keywords: alginate; carboxymethyl cellulose; nanofibrillated cellulose; human-derived skin fibroblasts; 3D cell-laden scaffolds; in vitro skin model 1. Introduction Skin, with its unique morphology, is critical for survival of the organism through acting as a functional barrier to a versatile range of environmental conditions, including stresses related to temperature changes, mechanical damage, and microorganisms [1–3]. Among its various vital functions, the human skin possesses highly effective self-regenerative properties, which is related to the presence of epidermal stem cells in different skin layers [1,4]. After damage, the skin activates its natural ability to promote spontaneous repair by following a four-stage process: hemostasis, inflammation, proliferation, and remodeling [4,5]. Loss of the epidermal barrier has serious adverse physiological effects. Open wounds present a high risk for fluid loss, high inflammation, bacterial colonization, and infection that can lead to sepsis. Nanomaterials 2020, 10, 733; doi:10.3390/nano10040733 www.mdpi.com/journal/nanomaterials Nanomaterials 2020, 10, 733 2 of 18 The reconstructed (e.g., scarred) tissue has a reduced functionality with loss of original properties of the uninjured skin [6]. Although a tremendous progress has been made over the past decades in the area of wound healing, autologous split-thickness skin grafts remain the gold standard in clinic for large wounds. However, there are several limitations of this approach. These include a shortage of donor skin tissue, as well as the potential additional health risks associated with the procedure (for example additional scarring, surgical site infections, hematoma, and bleeding) [7–9]. Lately, engineered skin substitutes present promising candidate products to alleviate these problems, and moreover, to induce a paradigm shift from using passive wound dressings to using bioactive cell-impregnated skin constructs [10]. Three-dimensional (3D) skin models have been under the spotlight for versatile applications in biomedicine, pharmaceutical, and cosmetic industries [3,10]. Beside skin grafts for wound healing, these have been proven as valuable in vitro models. Among others, they have been used to estimate skin permeability, to test potential adverse inflammatory responses that might occur during transdermal and topical drug invention, as well as served as important models to optimized drug formulations [11]. In contrast to animal skin, engineered skin exhibits several advantages, including better mimicking the human skin physiology, as well as mitigating ethical concerns and conforming to emerging regulations related to animal testing [11,12]. Furthermore, they serve as much better models (compared to animal models for example) to obtain fundamental insights into the etiology of skin diseases and related pathophysiological mechanisms associated with skin disease progression and treatment [13,14]. Although conventional tissue engineering methods have substantially contributed to clinical and dermatological studies, as well as facilitated the design and development of the first generation of skin grafts and models, at present, none of the bioprinted skin constructs can fully replicate native skin in terms of its morphological, biochemical, and physiological properties. However, simple skin constructs have already gained clinical value in burn and large wound treatment (e.g., Alloderm®, Biobrane®, Integra®)[3,7,10,11]. The inner micro- and macroscale features of the engineered skin tissue, which are prerequisite to accomplish satisfying aesthetic and functional results, have inspired scientists to employ additive manufacturing techniques that have already proven to be suitable for (basic) organ bioprinting and other tissue engineering strategies [3,10,11,15]. Bioprinting presents an advanced fabrication platform that enables preparation of 3D structured materials that allow a precise patterning of living cells and bioactive molecules (e.g., growth factors and cytokines) in predefined spatial locations [3,10,16]. By using computer-aided design (CAD), bioprinting has the potential to directly create graded macroscale architectures to better mimic the native extracellular matrix (ECM), therefore augmenting the attachment and proliferation of multiple types of cell concurrently. The 3D bioprinter has commonly the capability to dispense materials in X, Y, and Z directions, which facilitates the engineering of complex 3D structures in a bottom-up process. In addition, by utilizing multiple material platforms with various dispensing mechanisms, it enables incorporation of microfeatures to provide mechanical and biochemical cues at the microscale level that can further guide and enhance cellular alignment and differentiation. These characteristics assist the formation of intricate microenvironments and an architectural organization that can better satisfy various requirements of a natural niche for skin cells [10,16–18]. A critical step for (direct) bioprinting of a scaffold with incorporated cells lies in the selection and design of an ideal bioink. The latter must fulfill key requirements, including biocompatibility, desired viscoelastic behavior, a high mechanical integrity, and appropriate degradability. These properties are needed to achieve successful fabrication of complex biomimetic tissue substitutes. In addition, a bioink formulation also determines the selection of an appropriate bioprinting technique [18,19]. Typical bioinks incorporate a biocompatible hydrogel (bio)polymer solution and a suspension of desired cells. Hydrogels are appealing for cell encapsulation since they are mostly formed from polymers (e.g., polysaccharides, like alginate (ALG) and carboxymethyl cellulose (CMC)) that possess a favorable biocompatibility towards many cells. They provide structural support through direct contact with the Nanomaterials 2020, 10, 733 3 of 18 cells, mimicking the natural ECM environment, as well as govern the chemical and physical properties of bioinks [16,20]. Current skin cell-printing approaches mostly rely on sequential printing of dermal fibroblasts and epidermal keratinocytes encapsulated in a homogeneous bioink, usually made from ALG, collagen, and/or fibrin [3]. Over the past decade, type I collagen has prevailed over other natural polymers as the most used base ingredient in skin tissue engineering [3,21,22]. Nevertheless, many studies have shown that for wound healing purposes, polysaccharides ALG and CMC also have a similarly positive effect, which is related to their similarity in many aspects to the natural skin ECM. These similarities include a good mechanical strength and stiffness, biocompatibility, and providing a highly hydrated environment, favorable for the skin [23–27]. Still, the application of hydrogel-based bioinks in fabrication of clinically relevant 3D skin constructs can be challenging, which is mostly related to their suboptimal printability for this purpose. Diverse bioink synthesis techniques that employ